Relaxometric method for ex vivo characterization of tumor tissue, corresponding system and computer program product

Abstract

A method to assess presence of tumor tissue in a biological specimen resected during surgery / biopsy using the Field Cycling Relaxometry technique, system for carrying out the method of corresponding computer program product for controlling the system.

Description

[0001] "Relaxometric method for ex vivo characterization of tumor tissue, corresponding system and computer program product" **** FIELD OF THE INVENTION The invention concerns a method for the assessment of the presence of tumor tissue in a biological specimen resected during surgery / biopsy, preferably at the margins of the biological specimen, using relaxometric measurements. The invention also concerns a system for the assessment of the presence of tumor tissue in a biological specimen (i.e., a relaxometer) and a corresponding computer program product for controlling the system. BACKGROUND OF THE INVENTION In oncological surgery, the assessment of the absence of tumor cells in the outer regions of the resected tissues (the margins) is extremely important as it informs the surgeon about the complete removal of the tumor mass. In the case the presence of tumor cells at the outer surface of the resected specimen is detected (i.e., in the case of positive margins), this is taken as an indication of potential tumor recurrence at the site of the removed primary lesion, as well as an item suggesting that a metastatic process might already have taken place. Currently, information about the occurrence of positive / negative margins is acquired in the anatomopathology laboratory and it is available to the surgeon only a few days after the surgery. In the case of breast tumor conservative surgery, the occurrence of positive margins implies re-operation in 20-40% of the cases (i.e., a re-operation rate of about 20-40%), thus increasing the risks for the patients and the costs for the Health Care System. Access to the information about the negativity or positivity of the margins during the surgery is well recognized as an unmet medical need. As far as concern, the margins’ issue has been tackled with a number of physico-chemical techniques, namely: i. Microscopic analysis of frozen sections: the time required for microscopic analysis is compatible with the surgery but the method needs specialized personnel for the preparation and interpretation of the slides; sensitivity is around 83%; ii. Imprinting Cytology: the method provides for the surface of the tissue specimen pressed on a slide, fixed and colored. It relies on the fact that the malignant cells stick on the slide. It needs the assessment of an expert cytologist and has sensitivity around 72%; iii. X-ray imaging: these methods are usually characterized by a low sensitivity; iv. Magnetic Resonance Imaging: recently a scanner operating at 1 T has been proposed for the acquisition of images of the resected specimens with the Diffusion- Weighted Imaging (DWI) sequence. The method has good sensitivity (91%) and specificity (93%), but has two main drawbacks: the high cost and the need to have an expert radiologist for the interpretation of the DWI images; v. Ultrasounds: in general, the method sensitivity is highly variable (from 40% to 80%); moreover, specialized personnel in the surgery room is needed; vi. Nuclear Medicine approaches: they require the administration of radioactive tracers for targeting tumor cells before surgery. These methods have good sensitivity but the use of radioactive materials limits strongly their applicability; vii. Electromagnetic measurements: the method relies on the fact that physiological variations of the cells may alter the electromagnetic properties of the tissues. Sensitivity and specificity are around 70%. In the case of use of bioimpedance the sensitivity is reported to increase up to 84.3-87.35%; viii. Optical Imaging: the method relies on the fluorescent properties of inks used to color the sides of the resected tissue specimen. Interference with Hemoglobin can occur. Recently, administration of targeting dyes before surgery has been proposed, but its application results quite cumbersome; ix. Raman Spectroscopy: the method reports about the differences in the chemical composition between healthy and tumor cells. The acquisition of one measurement (one point) takes about 15 minutes. In conclusion, one may say that any of the above reported techniques has advantages and drawbacks. No technique owns properties that make it the technique of choice to tackle the margins’ issue. Kolodziejski K. et al. in the paper entitled "Markers of low field NMR relaxation features of tissues" published on Research Square (12 February 2024, pages 1- 17) disclose a method to assess presence of tumor tissue in a biological specimen resected during surgery / biopsy using the Field Cycling Relaxometry technique on the basis of the following criterion ^^ (^^ ) − ^^ (^^ )^^ = 1 1 1 2 wherein v1 = 1kHz and v2 = 10kHz are pre-defined (i.e. fixed) resonance frequencies at corresponding (fixed) measurement magnetic fields. Kolodziejski K. et al. state that a value of about 25-26% would be a suitablethreshold for parameterξto distinguish between tumorand healthy tissues. OBJECT AND SUMMARY OF THE INVENTION An object of the invention is to provide a method that allows to acquire highly reliable information on the presence of residual tumor tissue in a specimen resected during surgery / biopsy, preferably at the margins of the specimen, using the Field Cycling Relaxometry. Another object of the invention is to provide a system for carrying out the method that allows to acquire such information on the presence of residual tumor tissue. Another object of the invention is to provide a computer program product to control such a system. According to the invention, the above objectives are achieved thanks to the subject matter recalled specifically in the ensuing claims, which are understood as forming an integral part of this disclosure. The present invention concerns a method to assess the presence of tumor tissue in a biological specimen resected during surgery / biopsy using the Field Cycling Relaxometry technique (the flow diagram of the method being shown in Figure 8), the method comprising the following steps: a. obtaining a first sequence of at least m values of a measuring magnetic field strength Bri, wherein m ≥ 3, a second sequence of at least n values of a time τ, a detection magnetic field strength Bd, a re-cycle delay time RD, a polarization magnetic field strength Bp and a polarization time tp; b. exposing the specimen to a polarization magnetic field having a polarization magnetic field strength Bp for a time tp, thus allowing the magnetization build-up, if the current measuring magnetic field strength Bri is ≤ 7 MHz; c. exposing the specimen to a measuring magnetic field having a measuring magnetic field strength Bri for a time τ, thus allowing the specimen magnetisation to relax towards a magnetisation equilibrium value determined by the actual Bri; d. acquiring a value of magnetisation MZ of the specimen at time τ and at the measuring magnetic field strength Bri, the acquisition being carried out while exposing the specimen to a detection magnetic field having a detection magnetic field strength Bd after a 90° pulse has been applied to Mz; e. switching off the magnetic field for the re- cycle delay time RD; f. repeating steps b. to e. a number of cycles n, wherein the value of time τ varies at each cycle; g. fitting the values of magnetisation MZ acquired after n repetitions of steps b. to e. (magnetization intensity vs. τ) according to a monoexponential Bloch equation to produce a respective magnetization recovery / decay curve and calculate the proton longitudinal relaxation rate R1Briat the respective magnetic field strength Bri; h. repeating steps b. to g. a number of times m, wherein the value of the measuring magnetic field strength Bri varies each time, thereby calculating at least three proton longitudinal relaxation rates R1Br1, R1Br2, and R1Br3at the least three measuring magnetic field strengths Bri; i. calculating a parameter Slope according to the following formula: ^^ ^^^^2 ^^^^1 1 − ^^1^^ =^^^^^^3^ ^^^^3 ^^^^^^^^ 1^1^^^^2 − ^^^1^wherein Br3< Br2< Br1, and the parameter Slope exhibits one of a first positioning and a second positioning with respect to a cut-off; j. providing an indication of the presence of tumor tissue in the specimen if the parameter Slope has a first positioning with respect to the cut-off and an indication of the absence of tumor tissue in the specimen if the parameter Slope has a second positioning with respect to the cut-off. The present invention also concerns a system for carrying out the method and a corresponding computer program product for controlling the system. BRIEF DESCRIPTION OF THE DRAWINGS The invention will now be described in detail, purely by way of illustrative and non-limiting example, with reference to the attached figures, wherein: FIGURE 1. The Fast Field Cycling (FFC) experiment. A) Not-Polarized (NP) sequence (for high field strengths, i.e., usually Br> 0.17 T): the nuclear spin magnetisation is built up during the evolution period (τ) at relaxation field Br, then the nuclear magnetic resonance (NMR) signal is detected at detection field Bd. The sequence is repeated, staggering τ each time. B) Pre-Polarized (PP) sequence (for measurements at low field strengths, i.e., usually at Br< 0.17 T): the nuclear spin polarization is built up during the pre- polarization phase, at polarization field Bp. Relaxation occurs during the evolution period (τ) at relaxation field Br, then the NMR signal is detected at detection field Bd. The sequence is repeated, staggering τ each time. FIGURE 2. An example of the magnetisation decay and recovery curves obtained by applying the pre-polarized (PP) and non-polarized (NP) sequences, respectively. FIGURE 3. Block diagram of an FFC NMR relaxometer. FIGURE 4. Typical trend of the proton longitudinal relaxation rate R1 of human breast tissue as a function of the applied magnetic field: adipose (white dots) and tumor tissue (black dots). FIGURE 5. Schematic representation of an embodiment of the present invention applied to the assessment of margins of tumor resected specimens. FIGURE 6. Schematic representation of an embodiment of the present invention applied to the assessment of margins of breast tumor resected specimens. FIGURE 7. Example of the values that the parameter Slope and the cut-off can take for a breast healthy and tumor tissue. FIGURE 8. Flow chart of the method object of the present disclosure. Figure 9. Performance of different relaxometric classifiers on respect to the reference method (histopathological analysis) for positive and negative margin samples. On the right, the expression of the relaxometric classifier is shown, together with the related sensitivity (Sens.) and specificity (Spec.) values, for each A-C case, wherein case A corresponds to the Slope parameter according to the present invention, cases B and C correspond to the parameter ξ as defined in Kolodziejski K. et al. DETAILED DESCRIPTION OF THE INVENTION In the following description, numerous specific details are given to provide a thorough understanding of embodiments. The embodiments can be practiced without one or more of the specific details, or with other methods, components, materials, etc. In other instances, well-known structures, materials, or operations are not shown or described in detail to avoid obscuring aspects of the embodiments. Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. The headings provided herein are for convenience only and do not interpret the scope or meaning of the embodiments. The Field Cycling Relaxometry belongs to the field of the Nuclear Magnetic Resonance techniques, as the more known Magnetic Resonance Imaging. The Field Cycling Relaxometry allows access to magnetisation decay / recovery curves by acquiring the Free Induction Decay (FID) signal (corresponding to the magnetisation intensity) from the sample after exposure to a given magnetic field (measuring magnetic field Br, also indicated as relaxation magnetic field) for different intervals of time τ (see Figs. 1 and 2). By changing the intensity of the measuring magnetic field Br, proton longitudinal relaxation rates R1 can be determined at all frequencies permitted by the instrument and correlated to the chemical composition and dynamics of the proton containing species, mainly water, in the investigated tissue specimen. Figure 1 shows the typical acquisition sequences: the Not Polarized (NP) and Pre-Polarized (PP) sequences, for measurements at high (usually > 0.17 T) and low (usually < 0.17 T) field strengths, respectively. For the sake of clarity, 0.17 T corresponds to a proton Larmor frequency (= ^^B0, where ^ is the proton gyromagnetic ratio constant and B0 the magnetic field strength in Tesla) of 7 MHz. The PP sequence differs from the NP sequence because of the application of a high magnetic field (polarization magnetic field Bp) before the application of the relaxation field Br, in order to have a good signal intensity, otherwise not present at low fields. Therefore, in this type of experiment the magnetic field cycles between the polarization field (Bp) (if applied), several fields (Br) at which the proton relaxation time has to be measured, and the detection field (Bd). The detailed description of the experiment is reported in many papers [i.e., Ferrante G. and Sykora S.: Technical aspects of Fast Field Cycling, Advances in Inorganic Chemistry, 57, 405-470]. Briefly, the basic field cycling experiment with the NP sequence consists of the following steps (see fig. 1 – panel A): 1. The magnetic field is switched to the value of a measuring magnetic field Br for a time τ during which the magnetisation relaxes towards an equilibrium value, determined by the current Brvalue. 2. The magnetic field is switched to the value of a fixed detection magnetic field Bd and the equilibrium magnetisation established at Br is measured with a 90° pulse followed by acquisition of the magnetization recovery curve at Br. 3. The magnetic field is switched off for a certain time RD (Re-cycle Delay) that is the time between repetition of the cycles, equal to 0.5 - 1.5 s. The FID acquired at Bd (step 2) is proportional to the magnetisation at time τ at the given Br. The complete cycle should then be repeated a number n of cycles varying the value of τ (typically the number n of cycles of time τ is equal to 16 or 32, time τ ranging from 0.01 to 5 s in order to recover the entire magnetization recovery curve). Briefly, the basic field cycling experiment with the PP sequence consists of the following steps (see fig. 1 – panel B): 1. The sample is exposed to a high polarization field Bp for a polarization time tp with consequent building-up of the magnetisation of the sample. tp is selected in order to achieve a good signal intensity, i.e., with a signal to noise ratio (S / N) ≥ 3, preferably (S / N) ≥ 10. 2. The magnetic field is switched to the value of a measuring magnetic field Br for a time τ during which the magnetisation relaxes towards an equilibrium value, determined by the value of Br. 3. The magnetic field is switched to the value of a fixed detection magnetic field Bd and the equilibrium magnetisation established at Br is measured with a 90° pulse followed by the acquisition of the magnetization decay curve at Br. 4. The magnetic field is switched off for a certain Re-cycle Delay time RD that is the time between repetition of the cycles, equal to 0.5 - 1.5 s. The FID acquired at Bd (step 3) is proportional to the magnetisation at time τ at the given Br. The complete cycle should then be repeated a number n of cycles varying the value of τ (typically the number n of cycles of time τ is equal to 16 or 32 values, time τ ranging from 0.01 to 5 s in order to recover a large number of points covering the entire magnetisation decay curve). A graphical representation of a magnetisation decay / recovery curve (Intensity vs. τ) is provided in Figure 2. Each dot is obtained applying the steps previously described, which are repeated n = 32 times to obtain a decay / recovery curve. More precisely, the PP sequence was applied at a measuring magnetic field Br = 0.01 MHz with 32 values of τ logarithmically distributed in the range 0.0035-2.8 s; the NP sequence was applied at a measuring magnetic field Br = 10 MHz with 32 values of τ logarithmically distributed in the range 0.01-4 s. The fit of a magnetisation decay / recovery curve to an exponential function (Bloch equation) provides the proton longitudinal relaxation rate R1 (the reciprocal of the proton longitudinal relaxation time T1) at the field Br. In general, the Bloch equations are a set of macroscopic equations that are used to calculate the nuclear magnetisation M = (Mx, My, Mz) as a function of time τ when relaxation times T1 and T2 are present. These are phenomenological equations that were introduced by Felix Bloch in 1946 as disclosed, i.e., in F. Bloch, "Nuclear Induction", Physical Review 70, 4604–73 (1946). In the case of NP and PP sequences, the magnetisation recovery / decay curve refers to the z component of the nuclear spin magnetisation, Mz, towards its thermal equilibrium value (M0) at field Br, associated to the constant T1 (=1 / R1), i.e., the longitudinal (or spin-lattice) relaxation time, and, according to the Bloch equation, is defined by the following equations: −^^ ^^ ^^^^⁄^^^^^^ ^^ ^^ ^^ − ^^^^^^^^^^^^for the NP sequence and −^^ ^^^^ ^^ ^^^^^^ ∙ ^^⁄ ^^^^^^^^ 1^^^^^^^^for the PP sequence wherein ^^^^0^^^^and ^^^^0^^^^are parameters estimated from a fit procedure, where they are free to change as a function of the magnetic field strength Bri applied. ^^^^0^^^^corresponds to the highest measured intensity of the magnetization Mz at at each selected Bri. For the magnetization recovery curve acquired by the NP sequence, ^^^^0^^^^is the difference between zero and the Mz value at τ = 0; for the magnetization decay curve acquired by the PP sequence, ^^^^0^^^^is the difference between zero and the Mz value at τ → ∞ (see Fig. 2). The current commercial Fast Field Cycling relaxometers operate with a solenoid magnet which may achieve magnetic fields from few kHz to a maximum of 42 MHz in terms of proton Larmor frequency (i.e., to about 1 T). The block diagram of a typical system (i.e., an NMR relaxometer) is shown in Fig. 3. The NMR system includes a resistive solenoid (indicated as “Magnet” in Fig. 3) connected to a suitable magnet power supply unit (controlled via a dedicated magnet interface) and a cooling system, specifically designed for Field Cycling purposes. The NMR system further includes an electronic control unit that in turn includes an interface to the magnet power supply unit, a Variable Temperature Controller (VTC), a RadioFrequency (RF) unit (transmitter and receiver, including a preamplifier) and an acquisition unit. The electronic modulation of the current flowing through the coil of the electromagnet permits fast variations of the magnetic field induction and the acquisition of very short T1, at present, down to fractions of a millisecond. The probe, the RadioFrequency (RF) unit (transmitter and receiver, including a preamplifier), the sample Variable Temperature Controller (VTC) and the Acquisition Unit are conventional systems for NMR instruments, and a corresponding detailed description will not be provided herein for the sake of brevity. In particular, the acquisition unit detects the NMR signal from the sample and digitizes that signal for processing by a host computer system (or, more generally, a processing unit), e.g., a workstation or a computer system embedded in the relaxometer apparatus. A computer program (e.g., software package) running on the host computer system supports all experimental procedures (e.g., data acquisition, visualization and evaluation). The present invention relies on the observation of the present inventors that a tailored comparative procedure between magnetisation recovery / decay curves acquired at given Br values allows to assess the presence of tumor tissue in a resected specimen at the margins level. The intraoperative margin assessment according to the present invention is carried out in a short execution time and it is thus compatible with the surgical operation. The relaxation rate R1 of biological tissues is affected by the field strength at which the measurement is carried out. In particular, R1 shows a dramatic increase on going from high to low fields. At low fields the intracellular R1 is high (very short T1), whereas R1 of the extracellular compartment maintains a relatively low value. As water protons are the main source of the detected signal, the observed relaxation rates R1 are weighted to values that reflect either the ratio between the volumes of the two compartments and the exchange rate between them. They contain relevant information on the cell types present in the investigated tissue specimen and on the physio-pathological transformations occurring inside the cells and at the level of membrane water permeability. The exchange of water molecules across the tumor cellular membrane reflects either an enhanced metabolism (the higher production of metabolites implies an increase of the osmotic pressure) and the over-expression / up / down-regulation of transporters at the cellular membrane. In Fig. 4 the R1 values obtained from healthy breast tissue (white dots) and tumor breast tissue (black dots), in the field range corresponding to proton Larmor frequencies of 0.01 to 1 MHz, are reported. R1 of the healthy tissue results markedly higher than the ones of the tumor tissue. This behavior, characteristic of breast tissue, is due to the fact that in healthy tissue, with a large presence of adipocytes, there is a large contribution from fat molecules (which show very short T1) whereas in the tumor specimen, besides the decrease of adipocytes, there is an increase of the extracellular space and, at the same time, an increase of the water exchange rate between the intra- and extra-cellular compartments. In one embodiment, the present invention concerns a method to assess the presence of tumor tissue in a biological specimen resected during surgery / biopsy using the Field Cycling Relaxometry technique (the flow diagram of the method being shown in Figure 8), the method comprising the following steps: a. obtaining a first sequence of at least m values of a measuring magnetic field strength Bri, wherein m ≥ 3, a second sequence of at least n values of a time τ, a detection magnetic field strength Bd, a re-cycle delay time RD, a polarization magnetic field strength Bp and a polarization time tp; b. exposing the specimen to a polarization magnetic field having a polarization magnetic field strength Bp for a time tp, thus allowing the magnetization build-up, if the current measuring magnetic field strength Bri is ≤ 7 MHz; c. exposing the specimen to a measuring magnetic field having a measuring magnetic field strength Bri for a time τ, thus allowing the magnetisation of the specimen to relax towards a magnetisation equilibrium value determined by the actual Bri; d. acquiring a value of magnetisation MZ of the specimen at time τ and at the measuring magnetic field strength Bri, the acquisition being carried out while exposing the specimen to a detection magnetic field having a detection magnetic field strength Bd after application of a 90° pulse; e. switching off the magnetic field for the re- cycle delay time RD; f. repeating steps b. to e. a number of cycles n, wherein the value of time τ varies at each cycle; g. fitting the values of magnetisation MZ acquired after n repetitions of steps b. to e. (magnetization intensity vs. τ) according to a monoexponential Bloch equation to produce a respective magnetization recovery / decay curve and calculate the proton longitudinal relaxation rate R1Briat the respective magnetic field strength Bri; h. repeating steps b. to g. a number of times m, wherein the value of the measuring magnetic field strength Bri varies each time, thereby calculating at least three proton longitudinal relaxation rates R1Br1, R1Br2, and R1Br3at the least three measuring magnetic field strengths Bri; i. calculating a parameter Slope according to the following formula: ^^ ^^^^2 ^^ ^^^^1 1 −1^^^^ =^^^^^^3^^^^3 ^^^^^^ 1^^1^^^^2 − ^^^1^wherein Br3< Br2< Br1, and the parameter Slope exhibits one of a first positioning and a second positioning with respect to a cut-off; j. providing an indication of the presence of tumor tissue in the specimen if the parameter Slope has a first positioning with respect to the cut-off and an indication of the absence of tumor tissue in the specimen if the parameter Slope has a second positioning with respect to the cut-off. In one embodiment, the present invention concerns an NMR relaxometer system that includes: - a solenoid connected to a cooling system and a power supply unit, where the solenoid defines a space for hosting the probe in which the sample is settled; - a radio-frequency unit; - a magnet interface controlling the magnet power supply and thus the magnetic field generated by the solenoid; - a sample temperature control unit; - an acquisition module configured to acquire magnetisation data from the biological specimen; - a computer host equipped with a computer program (e.g., software package) for controlling the acquisition unit and controlling various steps of the experimental procedure (e.g., data acquisition, visualization and evaluation). In one embodiment, the present invention concerns a computer program product comprising instructions to operate an NMR relaxometer system according to the invention to execute the steps of the method according to the invention. The assessment of the optimal conditions of the Fast Field Cycling relaxometer will be acquired with an auto- test carried out before the start of the measurements' session using a properly designed phantom endowed with relaxation properties in the range of those expected for the investigated specimen. The phantom is made by a cross-linked protein in a buffered solution with a preservative agent and it is sealed under inert gas atmosphere. For example, the phantom consists of a solution of Bovine Serum Albumin (BSA) 9% in phosphate buffer pH 7.4 plus sodium azide 0.3 %, cross-linked by heat (20’ at 80°C) or by glutaraldehyde. The skilled man, with his common general knowledge, knows how to produce a phantom endowed with the relaxation properties in the range of those expected for the investigated biological tissue under analysis. The check of relaxometer performance consists of the acquisition of the R1 values at two magnetisation field strengths twice with the defined protocol. Both the signal intensity and the obtained R1 values are evaluated. The limits of acceptability for signal intensity, average and standard deviation values of the repeated R1 measurements have to be within the 5% of the values certified by the phantom manufacturer. The main parameters to be set for carrying out the method of the present invention are: - the number of times m and the values of the measuring magnetic field strengths Bri used for the acquisition of the magnetisation recovery / decay curves; - the value of the polarization magnetic field strength Bp and the polarization time tp; - the number of cycles n and the values of the time intervals τ for the acquisition of the magnetisation relaxation curves at each Bri value; - the waiting time before the starting of the new acquisition cycle (recycle delay, RD). In case the magnetisation Mz measured by the relaxometer is lower than a threshold value fixed by the instrument manufacturer, the method steps b. to h. are repeated at least one more time and the magnetisations Mz determined in the first scan are summed to the magnetisations Mz determined in the second scan. For determining the positivity / negativity of the biological sample (e.g., the margins of the resected tumor mass), the following parameters are calculated when carrying out the method: - the relaxation rate R1Briat a given Bri, obtained by the fit of the magnetisation recovery / decay curves (Intensity vs. τ) acquired by repeating steps b. to e., where the fit is carried out according to the monoexponential Bloch equation; and - the Slope, calculated as the slope of the straight line joining two normalized R1Brivalues measured at two different Bri where the normalization is done dividing the aforementioned R1Brivalues by a R1Brivalue measured at a third Bri according to the following equation, wherein Br3 < Br2 < Br1: ^^ ^^^^2 ^^ ^^^^1 1 −1^^^^^^3^^ ^^^^3 ^^^^^^^^^^ = 1 1^^^^2 − ^^^1^The presence of tumor tissue in the specimen is determined if the parameter Slope has a first positioning with respect to a cut-off and the absence of tumor tissue in the specimen is determined if the parameter Slope has a second positioning with respect to the cut-off. Figure 7 provides an example of the values that the parameter Slope and the cut-off can take for a breast healthy and tumor tissue. The parameter Slope of breast tumor tissue (positive margin) assumes a value (e.g., - 0.648) that is lower than the one (e.g., -0.299) of the breast healthy tissue (negative margin) (i.e., the slope of the straight line joining two R1Brivalues is higher for the tumor tissue than the healthy one). In such a case, the specimen contains tumor tissue if the parameter Slope is positioned below the cut-off (e.g., -0.517) and it does not contain tumor tissue if the parameter Slope is positioned above the cut-off. In other tissues / organs the situation can be the opposite, i.e., the parameter Slope of the tumor tissue assumes a value that is higher than the one of the healthy tissue, in such a case the specimen contains tumor tissue if the parameter Slope is positioned above the cut-off and it does not contain tumor tissue if the parameter Slope is positioned below the cut-off. The cut-off is determined in a training phase during which several resected organ / tissue tumor masses are subjected to the method disclosed above and then to the histological analysis provided by a surgical pathologist. The histological analysis (comprising tissue formalin-fixation, processing, sectioning, and staining with Hematoxylin & Eosin) is the reference method for tumor assessment and allows the correct distribution of the analyzed specimen in the positive or negative margin classes, i.e., the True positive (TP) and True Negative (TN) groups, respectively. The cut-off value to assign the sample to the correct class (i.e., containing or not tumor tissue) is evaluated according to the Receiver Operating Characteristic (ROC) curve. The ROC curve is constructed by plotting the true positive rate (TPR) against the false positive rate (FPR) for the different possible cut-off values. The true positive rate is the proportion of samples that are correctly predicted to be positive out of all positive samples (TP / (TP + FN)), where FN are the False Negative samples. Similarly, the false positive rate is the proportion of samples that are incorrectly predicted to be positive out of all negative samples (FP / (TN + FP), where FP are the False Positive samples. The TPR and FPR correspond to the sensitivity and the (1 - specificity) of the method, respectively. Each point on the ROC curve represents a sensitivity / specificity pair. The cut-off value is the point which classifies most of the samples correctly, i.e., it has the best sensitivity / specificity pair. In one embodiment, the acquisition is carried out at a detection magnetic field strength Bd comprised between 5 MHz and 20 MHz expressed in terms of proton Larmor frequency. In one embodiment, the measuring magnetic field strength Bri is comprised in the range 0.01 to 20 MHz in terms of proton Larmor frequency. In one embodiment, the relaxation rates R1Bri(=1 / T1Bri) are determined by applying the following Bloch equations: wherein ^^^^0^^^^is the highest measured magnetisation Mzat Bri; ^^^^0^^^^is the magnetisation Mzat τ = 0 if Bri> 7 MHz (i.e., the NP sequence) or the magnetisation Mzat τ → ∞ if Bri≤ 7 MHz (i.e., the PP sequence); and [1] is applied for Bri> 7 MHz (i.e., the NP sequence), equation [2] is applied for Bri ≤ 7 MHz (i.e., the PP sequence). In one embodiment, the measuring magnetic field strength Bri is set at least once (e.g., for m = 1) to a value higher than 0.3 MHz. In one embodiment, the measuring field magnetic strength Bri is set at least once (e.g., for m = 2) to a value lower than 0.3 MHz. In one embodiment, the specimen is a resected tumor mass having a weight comprised between 15 mg and 115 mg, preferably between 30 mg and 60 mg, as obtained by the surgeon in the surgery room. In one embodiment, the specimen is placed in a glass tube (having generally an outer diameter of 5 mm but other sizes are possible), the glass tube being introduced in the Field Cycling Relaxometer. In one embodiment, the steps d. to f. are carried out at a temperature ≤ 25 °C, preferably at about 10 °C. In one embodiment, when the steps d. to f. are carried out at a temperature lower than the room temperature (i.e., lower than 25 °C), the test tube containing the tissue specimen is brought to the selected temperature through the passage in a sample cooling device, wherein the sample cooling device is designed to host the test tube to allow the specimen to reach the desired temperature, preferably in less than one minute. In one embodiment, the polarization magnetic field strength Bp of the polarization magnetic field is comprised between 8 and 25 MHz. In one embodiment, the polarization step b. is carried out for a polarization time tp, wherein tp is comprised in the range 0.5 to 1.5 s. In one embodiment, the switching off of the magnetic field in step e. allows the specimen magnetisation to completely decay. In one embodiment, the switching off of the magnetic field in step e. lasts for at least 0.5 sec. In one embodiment, the number of cycles n ranges from 10 to 32. In one embodiment, the time value τ ranges from 0.01 to 4 sec. In one embodiment, in each cycle n, in which steps b. to e. are repeated, the time value τ is increased, preferably on a logarithmic basis. In one embodiment, the assessment of the presence of tumor tissue in the specimen involves evaluating the margins of the specimen. In the following some specific embodiments of the present invention are disclosed. Assessment of the presence of tumor tissue at the margins of resected specimens in tissue specimens The margin assessment protocol implies to apply the method disclosed above with the following specification: - Acquisition step: two acquisitions at a temperature ≤ 25 °C with protocol AP3 or AP4 (at Br< 0.05 MHz) and one acquisition with protocol AP1 or AP2 (at > 0.3 MHz) as reported in Figure 5. Protocols AP1 to AP4 are disclosed below. - Data analysis: the fit of the magnetisation recovery curves (Intensity vs. τ) obtained by repeating steps b. to e. is performed according to the monoexponential Bloch equation. This procedure allows the calculation of the R1Brivalues at the given Bri. The obtained R1Brivalues are used to calculate the normalized Slope. - The samples are classified looking at their Slope value in comparison with the cut-off value. Examples of settings for the R1 acquisitions at Br between 0.15 and 20 MHz are: − [AP1]: Bp = 9.5 MHz, tp = RD = 0.9 s, n = 14 τ distributed on a logarithmic basis in the interval 0.01-1.8 s; − [AP2]: Bp = 9.5 MHz, tp = RD = 0.9 s, n = 32 τ distributed on a logarithmic basis in the interval 0.01-4.0 s. Examples of settings for the R1 acquisitions at Br between 0.01 and 0.15 MHz are: − [AP3]: Bp = 9.5 MHz, tp = RD = 0.9 s, n =14 τ distributed on a logarithmic basis in the interval 0.0035-1.0 s; − [AP4]: Bp = 9.5 MHz, tp = RD = 0.9 s, n = 32 τ distributed on a logarithmic basis in the interval 0.035-2.8 s. Application of the method to assess the margins of breast tumors 127 small, freshly excised breast tissue samples (weight between 16 and 114 mg), belonging to 58 patients who underwent lumpectomy or mastectomy, were characterized from the relaxometric point of view according to the Figure 6, where the more general protocol M1 (Figure 5) is applied to breast cancer. In particular, the acquisitions were carried out at 10 °C and at: 0.01 and 0.02 MHz with the protocol AP4; and 1 MHz with the protocol AP2. The parameter Slope is calculated as follows: ^^ ^^^^^^ ^^^^^^ In this case, it was found that when the parameter Slope is greater than -0.517 (the cut-off), the sample is classified in the negative margin group. When the Slope is less than or equal to -0.517 the specimen is likely to contain tumor tissue thus suggesting the surgeon to remove more tissue from the area where the analyzed specimen was resected. The anatomopathologist's analysis provided the following results: specimens with Negative margins = 41 (S); specimens with Positive margins = 86 of which 39 classified as neat tumor and 47 as Mixtures of healthy cells and tumor cells. By comparing the classification obtained with protocol M1 and the anatomopathologist's analysis, 6 False Positive (FP) and 6 False Negative (FN) were found, thus yielding a sensitivity of 93%, specificity of 85% and accuracy (in terms of AUC, i.e., area under the curve) of 98.7%. The histological evaluation The histological analysis is the gold-standard reference and the results from the relaxometric determinations have been referred to the histological evaluation for all the investigated specimens. The typical procedure is as follows: Hematoxylin & Eosin (H&E) staining was conducted using standard methods on formalin-fixed, paraffin-embedded tissues. Samples were fixed in 10% neutral buffered formalin and embedded in paraffin. For histopathological analysis, samples were serially sectioned (5 μm) and every section stained with H&E. Histopathological scoring of tumor was performed by an expert anatomopathologist. Comparative example The article by Kolodziejski et al. (Research Square (2024), pages 1-17) proposes a relaxometric approach to differentiate between pathological and reference colon tissue. The following table summarizes the main differences between the method object of the present invention and the method disclosed by Kolodziejski et al. Table 1 The method Kolodziejski

[0002] The method Kolodziejski l i i l i t To demonstrate the advantage of a discriminant parameter containing three Br values as done in the present invention, the relaxometric data set used in the previous example (related to 127 small, freshly excised breast tissue samples) was analysed using the new parameters [p1] and [p2] given in the following equations, which are in best agreement with the parameters given in the article by Kolodziejski et al., taking into account the different range of magnetic field strengths probed.^^^^^^^^^^^ ^^^^ =^^^^^^2 − ^^^^^^1 ^^^^^^^^^^^^^ 1 1 obtained using the parameter Slope according to the present invention (case A) or the parameters given in the equations [p1] and [p2] (case B and C) according to Kolodziejski et al. As was the case for the Slope, the ROC analysis was employed to identify the most suitable cut-offs, which are displayed in the figure as lines (0.065 for [p1] and 25.96 for [p2], respectively). The parameters disclosed in Kolodziejski K. et al. fail to discriminate between samples with positive and negative margins in the way that the Slope parameter of the present invention does (sensitivity of 93%, specificity of 85%). In fact, the calculated sensitivity and specificity are 68.6% and 78% (9 FP, 27 FN) for [p1] (case B in figure 9), 80.2 % and 80.5 % (8 FP, 17 FN) for [p2] (case C in figure 9), respectively. In conclusion, the method object of the present invention is based on a protocol that can be applied to fresh tissue samples (intraoperative analysis allowed) using low-cost instrumentation. This approach demonstrates enhanced classification performance with respect to the results currently available according to the prior art.

Claims

CLAIMS 1. A method to assess presence of tumor tissue in a biological specimen resected during surgery / biopsy using the Field Cycling Relaxometry technique, the method comprising the following steps: a. obtaining a first sequence of at least m values of a measuring magnetic field strength Bri, wherein m ≥ 3, a second sequence of at least n values of a time τ, a detection magnetic field strength Bd, a re-cycle delay time RD, a polarization magnetic field strength Bpand a polarization time tp; b. exposing the specimen to a polarization magnetic field having a polarization magnetic field strength Bpfor a time tp, thus allowing the magnetization build-up, if the current measuring magnetic field strength Briis ≤ 7 MHz; c. exposing the specimen to a measuring magnetic field having a measuring magnetic field strength Brifor a time τ, thus allowing the specimen magnetisation to relax towards a magnetisation equilibrium value determined by the actual Bri; d. acquiring a value of magnetisation MZof the specimen at time τ and at the measuring magnetic field strength Bri, the acquisition being carried out while exposing the specimen to a detection magnetic field having a detection magnetic field strength Bdafter applying a 90° pulse; e. switching off the magnetic field for the re- cycle delay time RD; f. repeating steps b. to e. a number of cycles n, wherein the value of time τ varies at each cycle; g. fitting the values of magnetisation MZacquired after n repetitions of steps b. to e. according to a monoexponential Bloch equation to produce a respectivemagnetization recovery / decay curve and calculate the proton longitudinal relaxation rate R1Briat the respective magnetic field strength Bri; h. repeating steps b. to g. a number of times m, wherein the value of the measuring magnetic field strength Brivaries each time, thereby calculating at least three proton longitudinal relaxation rates R1Br1, R1Br2, and R1Br3at the at least three measuring magnetic field strengths Bri; i. calculating a parameter Slope according to the following formula: ^^ ^^^^2 ^^^^1 1 − ^^1^^^^wherein Br3< Br2< Br1, and the parameter Slope exhibits one of a first positioning and a second positioning with respect to a cut-off; j. providing an indication of the presence of tumor tissue in the specimen if the parameter Slope has a first positioning with respect to the cut-off and an indication of the absence of tumor tissue in the specimen if the parameter Slope has a second positioning with respect to the cut-off.

2. The method according to claim 1, wherein the detection magnetic field strength Bdis comprised between 5 MHz and 20 MHz expressed in terms of proton Larmor frequency.

3. The method according to any one of the preceding claims, wherein the measuring magnetic field strength Briis comprised in the range 0.01 to 20 MHz in terms of proton Larmor frequency.

4. The method according to any one of the precedingclaims, wherein the relaxation rates R1Bri(=1 / T1Bri) are determined by applying the following Bloch equations: −^^ ^^ ^^^^^^⁄ ^^^^^^^^ ^^ ^^ − ^^ 1 ^^^^^^^^Mzat Bri; ^^^^0^^^^is the magnetisation Mzat τ = 0 if Bri> 7 MHz or the magnetisation Mzat τ → ∞ if Bri≤ 7 MHz; and equation [1] is resolved for Bri> 7 MHz, equation [2] is resolved for Bri≤ 7 MHz.

5. The method according to any one of the preceding claims, wherein the measuring magnetic field strength Briis set at least once (e.g., for m = 1) to a value higher than 0.3 MHz.

6. The method according to any one of the preceding claims, wherein the measuring field magnetic strength Briis set at least once (e.g., for m = 2) to a value lower than 0.3 MHz.

7. The method according to any one of the preceding claims, wherein the steps d. to f. are carried out at a temperature ≤ 25 °C, preferably about 10 °C.

8. The method according to any one of the preceding claims, wherein the polarization magnetic field strength Bpof the polarization magnetic field is comprised between 8 MHz and 25 MHz.

9. The method according to any one of the preceding claims, wherein tpis comprised in the range 0.5 to 1.5 s.

10. The method according to any one of the preceding claims, wherein the switching off of the magnetic field in step e. lasts for at least 0.5 sec.

11. The method according to any one of the preceding claims, wherein the number of cycles n ranges from 10 to 32.

12. The method according to any one of the preceding claims, wherein the time value τ ranges from 0.01 to 4 sec.

13. The method according to any one of the preceding claims, wherein the assessment of the presence of tumor tissue in the specimen involves evaluating the margins of the specimen.

14. An NMR relaxometer system comprising: - a solenoid connected to a magnet power supply unit and to a cooling system, wherein the solenoid defines a space for hosting a probe in which a biological sample is insertable; - an electronic control unit that includes an interface coupled to the magnet power supply unit for controlling the power supply unit, a temperature controller coupled to the probe for controlling the temperature of the probe, a radiofrequency unit, and a data acquisition unit configured to acquire magnetisation data from the biological sample; and - a processing unit configured to control the dataacquisition unit to execute the steps of the method according to any one of claims 1 to 12.

15. A computer program product loadable in the memory of a processing unit of an NMR relaxometer system according to claim 13 and comprising instructions to cause the NMR relaxometer system to execute the steps of the method according to any one of claims 1 to 12.

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